Department of Pharmacology, CNR Institute of Neuroscience, University of Milan, Milan 20129, Italy.
J Biol Chem. 2011 Oct 7;286(40):34839-50. doi: 10.1074/jbc.M111.258384. Epub 2011 Jul 27.
Shank3/PROSAP2 gene mutations are associated with cognitive impairment ranging from mental retardation to autism. Shank3 is a large scaffold postsynaptic density protein implicated in dendritic spines and synapse formation; however, its specific functions have not been clearly demonstrated. We have used RNAi to knockdown Shank3 expression in neuronal cultures and showed that this treatment specifically reduced the synaptic expression of the metabotropic glutamate receptor 5 (mGluR5), but did not affect the expression of other major synaptic proteins. The functional consequence of Shank3 RNAi knockdown was impaired signaling via mGluR5, as shown by reduction in ERK1/2 and CREB phosphorylation induced by stimulation with (S)-3,5-dihydroxyphenylglycine (DHPG) as the agonist of mGluR5 receptors, impaired mGluR5-dependent synaptic plasticity (DHPG-induced long-term depression), and impaired mGluR5-dependent modulation of neural network activity. We also found morphological abnormalities in the structure of synapses (spine number, width, and length) and impaired glutamatergic synaptic transmission, as shown by reduction in the frequency of miniature excitatory postsynaptic currents (mEPSC). Notably, pharmacological augmentation of mGluR5 activity using 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)-benzamide as the positive allosteric modulator of these receptors restored mGluR5-dependent signaling (DHPG-induced phosphorylation of ERK1/2) and normalized the frequency of mEPSCs in Shank3-knocked down neurons. These data demonstrate that a deficit in mGluR5-mediated intracellular signaling in Shank3 knockdown neurons can be compensated by 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)-benzamide; this raises the possibility that pharmacological augmentation of mGluR5 activity represents a possible new therapeutic approach for patients with Shank3 mutations.
Shank3/PROSAP2 基因突变与认知障碍有关，从智力迟钝到自闭症不等。Shank3 是一种大型的突触后密度蛋白支架，参与树突棘和突触形成；然而，其具体功能尚未明确。我们使用 RNAi 敲低神经元培养物中的 Shank3 表达，结果表明这种处理特异性降低了代谢型谷氨酸受体 5 (mGluR5) 的突触表达，但不影响其他主要突触蛋白的表达。Shank3 RNAi 敲低的功能后果是 mGluR5 信号受损，如用代谢型谷氨酸受体 5 的激动剂 (S)-3,5-二羟基苯甘氨酸 (DHPG) 刺激后 ERK1/2 和 CREB 磷酸化减少所示，mGluR5 依赖的突触可塑性受损 (DHPG 诱导的长时程抑制)，以及 mGluR5 依赖的神经网络活动调节受损。我们还发现突触结构的形态异常 (棘突数量、宽度和长度) 和谷氨酸能突触传递受损，如微小兴奋性突触后电流 (mEPSC) 频率减少所示。值得注意的是，使用 3-氰基-N-(1,3-二苯基-1H-吡唑-5-基)苯甲酰胺作为这些受体的正变构调节剂，药理学增强 mGluR5 活性，恢复 Shank3 敲低神经元中 mGluR5 依赖的信号转导 (DHPG 诱导的 ERK1/2 磷酸化)，并使 Shank3 敲低神经元中 mEPSC 的频率正常化。这些数据表明，Shank3 敲低神经元中 mGluR5 介导的细胞内信号转导缺陷可以通过 3-氰基-N-(1,3-二苯基-1H-吡唑-5-基)苯甲酰胺来补偿；这提出了药理学增强 mGluR5 活性可能是 Shank3 突变患者的一种新的治疗方法。
